Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.     

Glycation free adduct accumulation in renal disease: the new AGE

Glycation adducts formed in the later stages of protein glycation reactions, advanced glycation endproducts (AGEs), are a class of uraemic toxin. Protein glycation was viewed originally as a post-translational modification that accumulated mostly on extracellular proteins. We now know that AGE residues are also formed on short-lived cellular and extracellular proteins. Cellular proteolysis forms AGE free adducts from these proteins, which are released into plasma for urinary excretion. AGE free adducts are also absorbed from food. AGE free adducts are the major molecular form by which AGEs are excreted in urine. They normally have high renal clearance, but this declines markedly in chronic renal failure patients, leading to profound increases in plasma AGE free adducts. Accumulation of plasma AGE free adducts is increased further in end stage renal disease patients on peritoneal dialysis and haemodialysis by increased AGE formation. The impact of AGEs absorbed from food is probably most marked for undialysed patients with mild uraemia. The toxicity of AGEs has been associated with resistance of the extracellular matrix to proteolysis and AGE receptor-mediated responses. AGE free adducts may also contribute to vascular disease in uraemia. They represent an important new age for glycation research in nephrology.     

终末期糖基化终产物通过转化生长因子依赖和非依赖途径介导NRK52E细胞转分化和胶原Ⅰ的合成

目的探讨终末期糖基化终产物（AGEs）介导肾小管上皮细胞转分化和胶原（Col）Ⅰ合成的分子机制。方法体外培养正常大鼠近端肾小管上皮细胞系（NRK52E），应用自制的AGE-牛血清白蛋白（BSA）刺激NRK52E细胞。免疫细胞化学方法检测不同时间磷酸化（P）Smad2／3核转位情况。ELISA方法检测细胞培养上清TGF-β1的水平。RT-PCR方法检测α-平滑肌肌动蛋白（SMA）、E-钙黏着糖蛋白（cadherin）和ColⅠmRNA表达。Western印迹检测α-SMA、E-cadherin和ColⅠ蛋白的表达。同时观察TGF-β1中和抗体对AGE-BSA上述效应的阻断作用。结果基础状态下，NRK52E细胞存在低水平p-Smad2／3核表达（16％）。与BSA对照组比较，AGE—BSA以时间依赖方式上调NRK52E细胞p-Smad2／3核转位，其高峰出现在30min（68％比30.5％，P〈0．01）和24h（76％比31．3％，P〈0．01）。AGE—BSA显著上调α-SMA和ColⅠmRNA和蛋白表达；下调E-cadherin mRNA和蛋白的表达；并能促进NRK52E细胞合成和分泌TGF-β1。TGF-β1中和抗体能明显抑制AGE—BSA介导的24hp-Smad2／3核转位（25．2％，P〈0．01），但不能阻抑30min活化高峰；能明显抑制AGE-BSA介导的α-SMA和ColⅠmRNA和蛋白表达．以及显著地上调E-cadherin mRNA和蛋白的表达。结论AGEs通过TGF-β依赖和非依赖途径诱导肾小管上皮细胞Smads信号通路活化，促进其向肌成纤维母细胞转分化和ColⅠ的合成。     

Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs) that are recognized by AGE receptors. Glyoxal, glycolaldehyde (GA), and methylglyoxal are potential intermediates for the formation of AGE structures such as Nomega-(carboxymethyl)lysine (CML). We evaluated the contribution of these aldehydes to the formation of AGE structure(s), particularly the structure important for the receptor-mediated endocytic uptake of AGE proteins by macrophages. GA-modified bovine serum albumin (BSA), methylglyoxal-modified BSA (MG-BSA), and glyoxal-modified BSA (GO-BSA) were prepared, and their physicochemical, immunological, and biologic properties were compared with those of glucose-derived AGE-BSA. CML contents were high in GO-BSA and low in GA-modified BSA (GA-BSA) but did not exist in MG-BSA. The fluorescence patterns of GA-BSA and MG-BSA were similar to those of glucose-derived AGE-BSA but were weak in GO-BSA. Immunochemically, the antibody against non-CML structures of glucose-derived AGE-BSA reacted strongly with GA-BSA and weakly with GO-BSA but did not react with MG-BSA. The negative charge of these ligands increased to a similar extent. However, GA-BSA, but not MG-BSA or GO-BSA, underwent receptor-mediated endocytosis by the macrophage-derived cell line RAW 264.7, which was effectively inhibited by glucose-derived AGE-BSA, acetylated LDL, and oxidized LDL, which are well-known ligands for the macrophage type I and type II class A scavenger receptors (MSR-A). The endocytic uptake of GA-BSA by mouse peritoneal macrophages was also significant, but that by peritoneal macrophages from MSR-A-deficient mice was markedly reduced. Our results suggest that GA serves as an important intermediate for the generation of AGE structure(s) responsible for recognition by MSR-A.     

Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells.

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.     

Evidence for Rho protein regulation of renal tubular epithelial cell function

BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton. The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known. METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells. RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis. A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis. A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells. Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis. The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor. CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis. Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.     

Highlights and Hotspots of Protein Glycation in End-Stage Renal Disease

Analysis of tissues, plasma, urine, other body fluids, and dialysate for glycation adducts has revealed the presence of two major forms: glycation adduct residues of proteins and related glycated amino acids—called glycation free adducts. The major effect on protein glycation in uremia is loss of clearance of glycation free adducts and their marked increase in plasma. Changes in glycation adduct residue content of plasma protein in uremia is, in contrast, relatively modest. There is now doubt as to whether the concept of interaction of advanced glycation endproduct (AGE)-modified proteins with putative AGE receptors can be sustained in vivo. A residual important feature of the receptor for AGEs may be decrease in expression of glyoxalase 1 of the antiglycation defence by S100A12 protein leaving the vasculature vulnerable to dicarbonyl stress and related AGE formation. The dicarbonyl proteome, proteins susceptible to dicarbonyl glycation at functional sites, is the likely mediator of glycation damage in uremia. Glycation of type IV collagen with shedding of endothelial cells and glycation of apolipoprotein B100 with increased atherogenicity of low density lipoprotein are two examples which may link protein glycation to increased risk of cardiovascular disease in end-stage renal disease.     

Autoantibody against N(epsilon)-(carboxymethyl)lysine: an advanced glycation end product of the Maillard reaction.

Prolonged incubation of proteins with reducing sugar produces advanced glycation end products (AGEs), which are implicated as factors for aging and diabetic complications. We previously demonstrated the presence of N(epsilon)-(carboxymethyl)lysine (CML), one of the main AGE structures, in human and animal tissues using a monoclonal anti-CML antibody (6D12). These findings suggest that CML structures present in vivo could serve as immunogens to generate autoantibodies. This suggestion was tested in the present study. First, plasma samples from diabetic rats reacted positively with AGE bovine serum albumin (BSA). These reactivities increased with the duration of diabetic states and were inhibited specifically by CML-BSA. Second, a fraction purified from plasma of diabetic patients, which bound to AGE-BSA, showed a positive reaction to CML-BSA and furthermore also to human lens proteins, which are known to undergo CML modification in vivo. Finally, patients with renal failure caused by diabetes or nondiabetic pathologies had a higher autoantibody activity against CML structure than that in normal subjects or diabetic patients without renal failure. These results indicate that CML accumulated in vivo serves as an immunological epitope to generate an autoantibody specific for CML that might be used as a potential marker for diabetic nephropathy or chronic renal failure.     

Targeted reduction of advanced glycation improves renal function in obesity

Obesity is highly prevalent in Western populations and is considered a risk factor for the development of renal impairment. Interventions that reduce the tissue burden of advanced glycation end-products (AGEs) have shown promise in stemming the progression of chronic disease. Here we tested if treatments that lower tissue AGE burden in patients and mice would improve obesity-related renal dysfunction. Overweight and obese individuals (body mass index (BMI) 26-39 kg/m(2)) were recruited to a randomized, crossover clinical trial involving 2 weeks each on a low- and a high-AGE-containing diet. Renal function and an inflammatory profile (monocyte chemoattractant protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF)) were improved following the low-AGE diet. Mechanisms of advanced glycation-related renal damage were investigated in a mouse model of obesity using the AGE-lowering pharmaceutical, alagebrium, and mice in which the receptor for AGE (RAGE) was deleted. Obesity, resulting from a diet high in both fat and AGE, caused renal impairment; however, treatment of the RAGE knockout mice with alagebrium improved urinary albumin excretion, creatinine clearance, the inflammatory profile, and renal oxidative stress. Alagebrium treatment, however, resulted in decreased weight gain and improved glycemic control compared with wild-type mice on a high-fat Western diet. Thus, targeted reduction of the advanced glycation pathway improved renal function in obesity.     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

Role of alpha(3)beta(1) integrin in tubulogenesis of Madin-Darby canine kidney cells

BACKGROUND: We isolated several Madin-Darby canine kidney (MDCK) subclones that exhibit different degrees of branching tubulogenesis in lower concentrations of collagen gel. The M634 clone formed cell aggregates in 0.3% collagen gel, but developed branching tubules vigorously in 0.1% collagen gel. In contrast, the Y224 clone formed cysts in 0.3% collagen gel and displayed fewer branching structures in 0.1% collagen gel. Morphologically, M634 cells exhibited higher levels of cell scattering as well as collagen-induced cell migration than Y224. We conducted this study to delineate the underlying mechanism of branching tubulogenesis in M634 cells. METHODS: Components of the focal contact machinery were analyzed in both cell lines, including the extracellular matrix glycoproteins fibronectin, laminin, and vitronectin; cytoskeleton-associated elements alpha-actinin, talin, and vinculin; and receptors for extracellular matrix and alpha(2), alpha(3), alpha(5), alpha(v), beta(1), and beta(3) integrins. Furthermore, we established several stable transfectants of alpha(3) integrin antisense RNA in M634 cells to examine the role of alpha(3)beta(1) integrin in branching morphogenesis directly. RESULTS: There were no obvious differences in levels of the focal adhesion complex proteins between M634 and Y224 cells, except that the content of the alpha(3) and beta1 integrins were 1.2- and 0.6-fold higher in M634 cells, respectively. The expression of alpha(3) integrin antisense RNA significantly lowered the levels of alpha(3) integrin mRNA and protein. The potential of cell scattering, migration, and branching tubulogenesis in M634 cells was inhibited according to the decrease in alpha(3) integrin expression. CONCLUSION: Our data indicate that expression of alpha(3)beta(1) integrin regulates cell scattering, migration, and branching tubulogenesis of MDCK cells, possibly via adhesion to or serving as a signaling molecule for type I collagen.     

Role of megalin in endocytosis of advanced glycation end products: implications for a novel protein binding to both megalin and advanced glycation end products

Advanced glycation end products (AGE) are filtered by glomeruli and reabsorbed and metabolized by proximal tubule cells (PTC). In renal failure, decreased renal AGE metabolism likely accounts for the accumulation in serum that is related to uremic complications. In diabetes, AGE generation is increased, and the handling mechanisms in PTC are likely associated with the pathogenesis of tubulointerstitial injury. It is therefore important to clarify the mechanisms of the AGE metabolism to develop a strategy for removing AGE in uremia and to elucidate the pathogenesis of diabetic nephropathy. To this end, this study focused on the molecular analysis of megalin, a multi-ligand endocytic receptor, in PTC. AGE uptake analysis was performed using the rat yolk sac-derived L2 cell line system established for the analysis of megalin's endocytic functions. The cells mediated specific internalization and degradation of AGE, which were significantly blocked by anti-megalin IgG, indicating that megalin is involved in the cellular processes. However, cell surface AGE-binding assays and ligand blot analysis revealed no evidence that megalin is a direct AGE receptor. Affinity chromatography and ligand blot analysis originally revealed that 200-kD and 400-kD proteins in the cells bind to AGE and the 200-kD protein to megalin in a Ca(2+)-dependent manner. The binding of megalin with the 200-kD protein was suppressed by receptor-associated protein (RAP), a ligand for megalin. In conclusion, megalin functions for endocytosis of AGE via an indirect mechanism. L2 cells express novel AGE-binding proteins, one of which may interact with megalin.     

晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响

目的：探讨晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响,以期揭示晚期糖化终末产物在骨质疏松发病中的作用。方法：用体外制备的AGE-牛血清白蛋白（AGE-BSA）加入人成骨细胞培养体系,观察不同浓度AGE和不同作用时间,对人成骨细胞增殖及分泌白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的影响。结果：加入AGE-BSA24h后,低浓度抑制成骨细胞增殖;培养48、72h后,浓度依赖性的促增殖作用增加。加入AGE-BSA48h,100μg/ml AGE-BSA促进成骨细胞分泌IL-6。200μg/ml-100μg/ml对IL-6及TNF-α的分泌均无显影响。结论：晚期糖化终末产物对人成骨细胞表现出在较高浓度时,仍具有很强的促增殖活性,提示人对高浓度AGE的耐受性增强。AGE在较高浓度对人成骨细胞分泌IL-6和TNF-α无显影响。提示由随龄增加的AGE在老年人引起的成骨细胞介导的骨吸收没有明显增加,与老年性骨质疏松为低转换型结果相一致。     

AGE, RAGE, and ROS in diabetic nephropathy

Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Two key mechanisms implicated in the development of diabetic nephropathy include advanced glycation and oxidative stress. Advanced glycation is the irreversible attachment of reducing sugars onto amino groups of proteins to form advanced glycation end products (AGEs). AGE modification of proteins may lead to alterations in normal function by inducing cross-linking of extracellular matrices. Intracellular formation of AGEs also can cause generalized cellular dysfunction. Furthermore, AGEs can mediate their effects via specific receptors, such as the receptor for AGE (RAGE), activating diverse signal transduction cascades and downstream pathways, including generation of reactive oxygen species (ROS). Oxidative stress occurs as a result of the imbalance between ROS production and antioxidant defenses. Sources of ROS include the mitochondria, auto-oxidation of glucose, and enzymatic pathways including nicotinamide adenine dinucleotide phosphate reduced (NAD[P]H) oxidase. Beyond the current treatments to treat diabetic complications such as the optimization of blood pressure and glycemic control, it is predicted that new therapies designed to target AGEs, including AGE formation inhibitors and cross-link breakers, as well as targeting ROS using novel highly specific antioxidants, will become part of the treatment regimen for diabetic renal disease.     

Radioreceptor assay for advanced glycosylation end products.

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1 mu CI/ml [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 micrograms/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid-BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycation of extracellular matrix proteins impairs migration of immune cells

The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes‐related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T‐cells. To achieve our purpose, we used in vitro AGE‐modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T‐cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin‐Alexa‐fluor 488‐labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE‐bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE‐modified matrix, but not with soluble AGEs like BSA‐AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs‐modified matrix protein inhibits cell migration and adhesion of Jurkat T‐cells.